1. Field of the Invention
The present invention relates to a carrier for immobilization of a microorganism for remediation of soil. The present invention also relates to a method for remediation of soil employing the carrier holding the soil-remedying microorganism.
The present invention further relates to a microorganism-immobilizing carrier for a bioreactor system, a biosensor, and an artificial internal organ utilizing the biological activity of an immobilized microorganism, in particular, to a microorganism-mobilizing carrier which is capable of exhibiting biological activity and improving survivability of the microorganism.
2. Related Background Art
In recent years, various noxious and hardly-decomposable chemical substances have been detected in soils, in rivers, in seas, and in the air, and aggravation of the pollution with such substances is attracting attention. In particular, soil pollution by organic chlorine compounds is causing serious problems. Establishment of techniques for preventing the spread of the pollution and for remedying the polluted environment is strongly desired. For example, soil pollution has become a matter of concern in gas production sites, oil refinery sites, demolished oil refinery sites, demolished fuel stockyard sites, demolished pulp plant sites, etc. Therefore development of soil remediation technique is strongly desired to clean the polluted soils.
The pollution of the soil not only prevents subsequent use of the land but also is liable to cause spreading of the polluted area by diffusion of the polluting substance into underground water and its diffusion with the water. Therefore a soil remediation technique is earnestly desired to be developed early.
Various soil remediation methods are known and have been tried to restore the soil to the original state by removing the polluting substance from the polluted soil.
The methods include physicochemical methods such as aspiration of the polluting substance from the soil by vacuum extraction. The physicochemical methods, however, involve many problems such as high cost, low operability, difficulty in treating the polluting substance existing at a low concentration.
On the other hand, soil remediation utilizing a microorganism, namely bioremediation, is promising.
The bioremediation methods include strengthening of self-cleaning function of ecosystem by activating a native microorganism in the soil to decompose the polluting substance into an innoxious substance; and include also, as an improved method, intentional introduction of microorganism having the ability to decompose the polluting substance from the outside to accelerate the remediation of the polluted soil.
Many kinds of microorganisms are known which have ability of decomposing the polluting substances detected in the soils. Direct application of such a microorganism to the soil does not give usually the intended cleaning effect because the applied microorganism individuals rapidly decrease in number in the soil in a short period.
To solve the problem of rapid decay of the applied microorganism in the soil, a large amount of nutrients for the applied microorganism may be introduced to the soil. This requires a high cost and may cause secondary pollution with the nutrients. Therefore, a method is demanded for maintaining the remediation activity by prolonging the life of the microorganism in the soil without supplying a large amount of the nutrients.
The change of the number of microorganism cells applied to the soil is greatly affected by the water content and the nutrient content of the soil. The amounts of water and the nutrients in the soil depend on the location of the soil, the landform, the vegetation on the soil, the depth, and the soil composition. These factors greatly affect the physiological activity of the microorganism growing in the soil. The situation is the same in the polluted soil.
For example, if the water content of the soil, more correctly the content of water available to the applied microorganism, is not in the range suitable for the microorganism, the applied microorganism usually does not grow well or does not manifest satisfactory activity. If the water content in the environment is deficient, the activity of the microorganism drops: a decomposing microorganism decreases its decomposition activity. At further deficiency of the water, a microorganism having no drought-resistance decreases in number and finally dies, whereas a drought-resistant microorganism also forms spores or goes into a dormant state to exhibit extremely low activity, whereby the decomposition of the noxious substance is not achieved. On the contrary, if the water in the environment is excessive, the oxygen content in the water becomes deficient, and an aerobic microorganism decreases its activity or decreases the number of microorganism cells. Since most of the microorganism having high activity in decomposing polluting substances are aerobic, the oxygen-deficiency is usually undesirable.
Another cause of decrease of the microorganisms in the soil is competition between the applied microorganism and native microorganisms, particularly predation of applied microorganism by protozoa. Suppression of the predation is necessary to improve the survivability of the applied microorganism.
On the other hand, in addition to the improvement of survivability of the applied microorganism, the bioremediation involves another problem on efficiency of diffusion of the applied microorganism in the soil. The bioremediation is directed to a polluted land over a large area which contains a pollutant at a low concentration, and therefore cannot be treated by a physicochemical method such as vacuum extraction. To clean such soil practically, the decomposing microorganism is required to diffuse in the polluted soil. However, the microorganism cannot easily migrate in the soil. Therefore, a method needs to be developed for bringing the microorganism to the proximity to the polluting substance in the soil. Currently conducted methods include injection of a decomposing microorganism into soil by pressure of water or air, application of a large amount of a decomposing microorganism, and so forth. With such a method, however, the diffusion of the microorganism is extremely impeded in some kinds of soils such as soils of a high clay content or of a low water content. Therefore, an improved method is required.
Some pollutant-decomposing microorganisms require coexistence of a low molecular weight compound called an inducer that induces expression of the enzyme. For example, Methylocystis sp. M strain does not decompose TCE (trichloroethylene) until the methane monooxygenase activity is induced by the presence of methane. Pseudomonas cepacia KK01 strain (Deposit No. FERM BP-4235) described in Japanese Patent Application No. 4-103180 decomposes TCE by appearance of toluene-monooxygenase induced by the presence of phenol.
As described above, the biodegradation of noxious substance in the soil or underground water from the polluted soil is conducted usually by a specific decomposing microorganism by co-oxidation or a like mechanism. Therefore, methods are tested or considered which introduce the required substance such as methane or phenol from the outside into the soil. Such methods, however, not only accompany other danger (e.g., inflammation, pollution, etc.) caused by the introduced substance but also cause diffusion of the introduced substance into a larger area or into the air or retention of the substance for a long term by adsorption by soil, which are therefore not practicable.
A method is studied in which the microorganism is mutated by gene technology so as not to require any inducer. In the open system for soil remediation, however, confirmation of safety and legal control of this method are not sufficient and thus, the method is not practicable at present.
Production of useful materials or treatment of waste materials by use of biological activity are widely conducted as a bioreactor. The employed organism includes prokaryote, eukaryote, and recombinant thereof, and is selected therefrom depending on the object. Immobilization of the microorganism in a high density is effective to mitigate mechanical shocks to the organism, to conduct recovery of the product efficiently, and to conduct the treatment continuously. The microorganism-immobilizing carrier investigated for such purposes includes polysaccharides, agarose, alginate gels, acrylamide gels, collagen, and synthetic material modified chemically to have increased suitability for organisms. The method of immobilization of the microorganism includes incorporation by solidification of a polymer, polymerization, or chemical crosslinking with a bonding agent.
The nutrients for growth need to be supplied to the immobilized microorganism by diffusion from outside the immobilizing carrier such as immersion of the microorganism-immobilizing carrier in a liquid culture medium.
In order to maintain the activity of the immobilized microorganism for a long term, an inducer and minerals have to be added by diffusion from the outside in the same manner as the aforementioned nutrients. The inducer is required to be supplied constantly because it stimulates the immobilized microorganism to cause biosynthesis of a specific enzyme system to manifest the activity. However, simple constant supply of the inducer is not sufficient, but the inducer is required to be supplied so as to keep an optimum concentration, because some inducers at a high concentration are harmful to microorganisms to inhibit growth of the cells, and affect adversely the natural environment including humans. For example, a certain Pseudomonas bacterium requires toluene, phenol, or cresol as the inducer for biological decomposition of TCE, with an optimum concentration of the inducer. Naturally an insufficient amount of the inducer does not initiate the activity, while an excessive amount of the inducer inhibits growth of the bacteria. Therefore, the inducer needs to be supplied constantly to keep its optimum concentration for a long period.
However, the introduction of the inducer like phenol and toluene from the outside of the immobilizing carrier is limited since it causes pollution outside the carrier. Because of the low utilization efficiency of the introduced inducer, the amount of the introduction is inevitably larger, which amplifies the pollution. Moreover, the constant supply at the optimum concentration range is extremely difficult. Therefore, use of a bioreactor which employs a microorganism requiring an inducer is restricted greatly. Such problems are not limited to the supply of inducers, but are involved in supply of nutrients and growth factors.